Journal: bioRxiv
Article Title: Single-Cell Spatial Mapping of Human Kidney Development Reveals the Critical Role of the Local Microenvironment in Cell Fate Decisions
doi: 10.1101/2025.06.15.659579
Figure Lengend Snippet: ( a ) Spatial locations of CytoSignal ligand scores for IGF1 and IGF2 in the human kidney, with score indicated by color (low being purple and high being yellow). ( b ) Histogram of correlations of microenvironment score with absorption probability for 878 microenvironment signals for the renewal trajectory. X-axis shows correlations and y-axis shows frequency (density estimate) of that correlation. Some individual genes of interest are labeled. ( c ) Ligand score (y-axis) is plotted against pseudotime (x-axis) for IGF2. Each trajectory is shown by its discrete color. ( d ) UMAP of scRNAseq showing That IGF2 is most expressed in the NPC cell population, with cognate receptors IGF1R and INSR expressed in all cell populations. ( e ) EpCam immunofluorescence staining (white: ureteric tree) images and quantified number of UB tips (y-axis) for mouse fetal explants incubated with ectopic IGF2 ligand. Total number of tips was normalized for average number of tips in the control samples for each replicate. This was performed in 3 separate litters. Scale bar = 400 um.} ( f ) EpCam immunofluorescence staining (white: ureteric tree) images and quantified number of UB tips (y-axis) for mouse fetal explants incubated with ectopic insulin ligand. Scale bar = 400 um. This experiment was repeated in a separate mouse litter. Total number of tips was normalized for average number of tips in the control samples for each replicate. This was performed in 3 separate litters. Scale bar = 400 um. Each replicate shown separately in Supplementary Fig. 15. ( g ) SIX2 immunofluorescence staining (white: NPC marker) images and quantified size of SIX2 caps. Area was normalized to average area for control explants for each 3 kidneys per condition performed in 2 separate litters (12 total kidneys). Scale bar = 400 um. Each replicate shown separately in Supplementary Fig. 15. ( h ) SIX2 (purple) and EpCam (white) immunofluorescence of explants treated with IGF1R inhibitor linsitinib at various concentrations. This was repeated in a separate litter and had similar results. Scale bar = 400 um. Each replicate shown separately in Supplementary Fig. 15. ( i ) Phase, SIX2 and JAG1 immunofluorescence of human kidney organoids treated with IGF1R inhibitor linsitinib at various concentrations. This experiment was repeated with similar results in a separate organoid batch. Scale bar indicates 1000 um. ( i ) Histogram of correlations of microenvironment score with absorption probability for 878 microenvironment signals for the PT trajectory. X-axis shows correlations and y-axis shows frequency (density estimate) of that correlation. Some individual genes of interest are labeled. ( j ) Spatial locations of CytoSignal ligand scores for AVP in the human kidney, with score indicated by color (low being purple and high being yellow). ( k ) UMAP of scRNAseq showing That AVP is most expressed in the PT cell population, with cognate receptors AVPR1A and AVP2R expressed in the DCT cell population. ( l ) Ligand score (y-axis) is plotted against pseudotime (x-axis) for AVP. DCT and PT trajectory are shown by their own discrete color. ( o ) CosMx cell type annotations of renal corpuscles, with PT, podocyte and PEC annotations shown in space. ( p ) Immunofluorescence of human fetal kidney tissue showing ACE2 expression (purple), WT1 expression (green) and DAPI (white) ( q ) UMAP showing probability of trajectories for renal corpuscle and tubular fate indicated for each cell based on color (left). Histogram of ligand correlations with renal corpuscle fate within the PT population. X-axis shows correlations and y-axis shows frequency (density estimate) of that correlation. Individual genes of interest are labeled. Scale bar = 100 uM. ( r ) Spatial locations of CytoSignal ligand scores for AVP in the human kidney, with score indicated by color (low being purple and high being yellow). ( s ) UMAP of scRNAseq showing That AGT, and ACE2 are expressed in the PT cell population, with cognate receptor AGTR2 expressed in all NPC derived cell types. PT: proximal tubule, LOH: loop of Henle, DCT: distal convoluted tubule, PEC: parietal epithelial cell, NPC: Nephron progenitor cell, Int: intermediate. IGF2: insulin like growth factor 2, AVP: arginine vasopressin, IGF1: insulin like growth factor 2. AVP: Arginine vasopressin. AGT: Angiotensinogen, ACE2: Angiotensin converting enzyme 2, AGT2R: Angiotensin 2 receptor, AVPR1A: arginine vasopressin receptor 1a, AVPR2: arginine vasopressin receptor 2. MsE: mouse explant. HumSpatial: Human Spatial, HumOrg: Human organoids, HumIF: human immunofluorescence.
Article Snippet: After fixation, explants were blocked in 5% donkey serum and then stained with the following primary antibodies prior to imaging, JAG1 - goat (1:200), (R&D, AF599-SP), Six2 - mouse (1:100) (proteintech, 66347-1-Ig) Six2 – rabbit (1:200) (proteintech 11562-1), LTL (1:200) (Vector Laboratories B-1325), ECAD (1:200) (abcam, ab11512), NPHS1- (R&D, AF4269) sheep (1:200), ITGA8 (1:200) (R&D, AF4076).
Techniques: Labeling, Immunofluorescence, Staining, Incubation, Control, Marker, Expressing, Derivative Assay
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